Research progress and ecological function of phages in soil
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摘要:
噬菌体在地球表层生态系统中的元素循环及污染物转化的微生物群落调控中发挥重要作用。相比于水生系统，土壤高度的异质性和病毒颗粒的高吸附度给土壤中噬菌体的研究带来挑战，尚未引起足够的关注。本文介绍了土壤噬菌体的形态与生命周期分类、以及噬菌体的提取和分析方法；探讨噬菌体调控土壤微生物群落结构以及碳、氮、磷、硫等生命元素循环与重(类)金属转化的科学联系；揭示其生物地球化学机制，阐明其生态功能和环境意义；并对土壤中噬菌体未来研究重点进行了展望。
Abstract:Phages play important roles in modulating microbial communities, and subsequently determining element circulation and pollutant transformation in the earth surface ecosystem. Compared with aquatic systems, the high heterogeneity of soil and the high adsorption of virus-like particles bring great challenges to the study of phages in soil, and leading to insufficient attention by far. In this review, the morphological and life cycle classification, extraction and analytic methods for phages in soil, were briefly summarized. In addition, the scientific linkage of phages in regulating soil microbial community structure and the cycle of life elements (e.g. carbon, nitrogen, phosphorus and sulfur) with the transformation of heavy metals (metalloid), were further discussed. The underlying biogeochemical mechanisms were revealed, and the ecological functions and environmental significances were clarified. Finally, the future research focuses of soil phages were prospected.
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铁皮石斛Dendrobium officinale是兰科Orchidaceae石斛属Dendrobium多年附生草本，以新鲜或干燥的茎入药。现代研究发现铁皮石斛含有多糖、生物碱及酚类等多种有效活性成分，其提取物具有抗氧化、降血糖及防治糖尿病并发症、抗肿瘤、提高免疫力等多种药理作用[1-2]。铁皮石斛作为药食同源药材，已被大量应用于疾病及日常生活保健中，其需求量急剧上升[3-4]。目前铁皮石斛种子、苗茎段、原球茎等是市场规模化培养常用材料来源，在一定程度上解决了铁皮石斛资源严重短缺的问题[5]。但是人工种植铁皮石斛仍存在以下问题：试管苗移栽成活率偏低、生长周期长，致使铁皮石斛种植成本高而产量低[6]；人工种植铁皮石斛质量参差不齐，且种植铁皮石斛有效成分含量相对低于野生铁皮石斛植株[7-8]；人工密集种植导致铁皮石斛较易遭受微生物病害的侵染，造成炭疽病、白绢病、黑斑病、黑腐病等石斛病害的发生[9]，这些现象被认为与组织培养苗生产与传代过程中缺乏植物内生菌有密切关系[10]。
植物内生菌是指那些在生活史的一定阶段或全部阶段生活于植物的各种组织和器官内部并与宿主植物在长期共同进化过程中形成共生关系的一类微生物[11]。作为兰科植物，铁皮石斛的种子虽然数量众多但没有胚乳，在自然条件下必须与微生物建立共生关系才能完成生活史[12]。目前普遍认为，石斛内生菌可以通过增强水分和矿质营养吸收，产生生长调节剂和维生素等物质以促进植株根、茎、叶的生长及提高活性成分[13]。
石斛内生菌是影响石斛生长与药效的重要生态因素之一。目前石斛内生菌的研究主要集中在真菌方面，细菌作为内生菌重要组成部分和活性类群研究较少。比较野生和人工铁皮石斛内生细菌结构差异，研究野生铁皮石斛内生细菌活性和在组培苗中的定殖情况，对于揭示植物内生菌结构形成的驱动因素、增强铁皮石斛驯化苗对自然环境的栽培适应性、提高铁皮石斛产量具有重要意义。
1. 材料与方法
1.1 药用植株和菌株
野生铁皮石斛采自广东省清远市(23°20′49″N, 113°12′ 11″E), 株高10~15 cm；人工栽培铁皮石斛采自广东省潮州市饶平铁皮石斛人工种植基地(23°10′49″N，113°21′9″E)，株高20~35 cm。石斛样品生长状态均良好。采样时将整株植物完整挖出，无菌采样袋密封好放入冰盒，运送至实验室后置于4 ℃冰箱中保存, 在2 h内进行内生细菌的分离。使用的铁皮石斛炭疽病致病菌为胶孢炭疽菌Colletotrichum gloeosporioides，由广州中医药大学中药学院张桂芳老师馈赠。
1.2 培养基和试剂
马铃薯葡萄糖琼脂(PDA) 培养基购于广东环凯微生物科技有限公司。活性菌筛选培养基配制参考文献[14]。2×T5 Direct PCR kit(Tsingke) 购于北京擎科新业生物技术有限公司。共生培养基配制：1/2 MS(购自海博生物技术有限公司)+2 g/L活性炭+8 g/L琼脂。引物27F(5′-AGAGTTTGATCCTGGCTCAG-3′)和1492R(5′-GGTTACCTTGTTACGACTT-3′)由深圳华大基因股份有限公司合成。
1.3 内生细菌的分离和纯化
用自来水冲洗掉铁皮石斛植株上的泥土后，将其根、茎、叶分开，剪成约2 cm的小段, 依次用φ为75% 的乙醇溶液浸泡2 min，10.91~65.45 g/L的NaClO溶液浸泡5 min，无菌水冲洗至少3遍, 用无菌镊子和剪刀将植株根、茎、叶剪碎，取0.1 g剪碎组织置于无菌研钵中，加无菌水1 mL研磨成匀浆，取0.1 mL组织匀浆均匀涂布于PDA平板上，37 ℃条件下培养14 d左右，每天观察菌株生长状况，将新长出菌落转接新的PDA平板，根据肉眼可辨的菌落形态特征，将从同一器官内分离到的相同菌株去除，转接纯化至单菌落后保存。同时收集表面消毒时最后1次冲洗样品的水样，以同样的方法涂布于PDA平板检测消毒效果。
1.4 内生细菌的鉴定
使用Direct PCR kit扩增细菌16S rDNA。挑取单菌落菌体加入50 μL Lysis buffer A中，95 ℃水浴10 min，加入等量Lysis buffer B作为缓冲液；涡旋振荡后短暂离心，取上清液作为DNA模板，以27F和1492R为引物扩增细菌16S rDNA，PCR反应条件:98 ℃预变性2 min；98 ℃变性10 s，55 ℃退火20 s，72 ℃延伸30 s，共30个循环；72 ℃终延伸2 min。PCR产物经琼脂糖凝胶电泳鉴定无误后送上海美吉生物有限公司测序，测序序列于Blast( https://blast.ncbi.nlm.nih.gov/blast.cgi) 中比对。选取代表性菌株的16S rDNA序列信息，使用MEGA7.0软件，采用邻接法构建其系统发育树，并用iTOL进一步美化。
1.5 内生细菌活性筛选
参照文献[14]的方法筛选解磷、解钾、产IAA和固氮的活性菌株。参照文献[15]的方法筛选产铁载体的活性菌株。将筛选菌株和致病炭疽菌接种在PDA平板上,两菌相距5 cm，置于培养箱内28 ℃条件下培养3~5 d，观察并记录菌株生长情况和菌株间的拮抗现象。每种拮抗菌设3次重复。
1.6 内生细菌定殖动态
将内生菌于37 ℃、250 r/min条件下培养至对数期，稀释成1×106 CFU/mL菌液备用。随机挑选健康且长势一致(2~4叶期)铁皮石斛组培苗，移至含有共生培养基的旋口瓶中，每瓶3棵苗。吸取0.5 mL稀释菌液添加至试管苗根部，对照组仅添加0.5 mL培养基。将试管苗置于温度(25±2) ℃、光照度1 500 lx、每天光照时间12 h、空气相对湿度60%~80% 的条件下培养。分别取接种后3、10、20和30 d的铁皮石斛苗，用无菌剪刀将其根、茎、叶分开，按“1.3”方法表面消毒以及检测消毒效果。将根、茎和叶分别置于无菌的研钵中研磨至匀浆，再用无菌蒸馏水梯度稀释，涂布平板，于37 ℃条件下培养48 h后，统计回接菌定殖情况。
1.7 数据处理
参考文献[16]的方法对野生和人工栽培铁皮石斛内生细菌的Shannon-Wiener多样性指数(Shannon-Wiener diversity index，H′)、均匀度指数(Evenness index，E)、相似性系数(Similarity coefficient，C) 进行计算及分析。采用Excel、SPSS 软件进行数据整理统计，利用Duncan’s 法分析铁皮石斛不同部位的多样性指数差异, 采用t检验分析不同生境下的数据是否存在差异。
2. 结果与分析
2.1 野生与人工栽培铁皮石斛内生细菌的类群结构
从野生铁皮石斛植株中分离到217株内生细菌，根据测序比对和系统发育分析结果，内生细菌可以归类为芽孢杆菌属Bacillus、微杆菌属Microbacterium、不动杆菌属Acinetobacter、短食单胞菌属Stenotrophomonas、硫胺素芽孢杆菌属Aneurinibacillus、棒状杆菌属Leifsonia、短小杆菌属Curtobacterium、无色菌属Achromobacter、泛菌属Pantoea等9个属(隶属于3个门)(图1)。其中芽孢杆菌属和不动杆菌属菌株数分别占总分离菌株数的79.26%和8.76%，为优势菌群。野生铁皮石斛不同组织内生细菌类群结构存在差异(图2)。分离自根的内生细菌有70株，优势菌群是芽孢杆菌属(占根部分离菌株的81.16%)、微杆菌属(7.25%)和不动杆菌属(5.8%)。来自茎的内生细菌有128株，优势菌群是芽孢杆菌属(占茎部分离菌株的79.84%)、不动杆菌属(9.3%)和短食单胞菌属(6.2%)。叶中分离到19株细菌，以芽孢杆菌属(占叶部分离菌株的68.42%)、不动杆菌属(15.79%)为优势菌群。芽孢杆菌属、不动杆菌属、短食单胞菌属存在于野生铁皮石斛根、茎、叶各组织部位。
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图 1 基于16S rDNA建立的部分铁皮石斛内生细菌系统发育树Figure 1. Phylogenetic tree based on 16S rDNA gene sequence of partial endophytic bacteria isolated from the Dendrobium officinal![]()
图 2 野生铁皮石斛可培养内生细菌在属水平的相对丰度Figure 2. The relative abundance of cultivatable endophytic bacteria isolated from wild Dendrobium officinale at the genus level从人工栽培铁皮石斛植株中分离到68株内生细菌，归类为伯克霍尔德菌属Burkholderia、埃希菌属Escherichia、泛菌属Pantoea 3个属(隶属于1个门)(图1)。其中伯克霍尔德菌属和埃希菌属分别占总分离菌株数的54.41%和30.88%，为优势菌群。人工栽培铁皮石斛不同组织内生细菌类群结构存在差异（图3）。根中分离到细菌34株，茎中分离到细菌22株，均以伯克霍尔德菌属(占根部分离菌株的62.5%，占茎部分离菌株的60.87%)和埃希菌属(占根部分离菌株的37.5%，占茎部分离菌株的30.43%)为优势菌群，并且这2个菌群也存在于叶中。叶中分离到细菌12株，以泛菌属(61.54%)为优势菌群。
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图 3 人工栽培铁皮石斛可培养内生细菌在属水平的相对丰度Figure 3. The relative abundance of cultivatable endophytic bacteria isolated from artificial cultivation Dendrobium officinale at the genus level以上结果表明，野生与人工栽培铁皮石斛内生菌类群和比例存在较大差异，只有泛菌属在野生和人工栽培铁皮石斛中都可以分离到。
2.2 野生与人工栽培铁皮石斛内生细菌的多样性和相似性
从多样性指数(H′)来看(表1)，野生铁皮石斛内生细菌的H′为0.85，根、茎、叶的内生细菌H′分别为0.75、0.80和1.02，根、茎、叶内生细菌的均匀度指数(E)分别为0.42、0.36、0.63，叶中内生细菌的H′和E显著高于根和茎中的相关指数。野生植株中茎与根的相似性系数(C)最高(C茎−根=0.72)，其次是茎与叶(C叶−茎=0.65)，根与叶的相似性最低(C根−叶=0.60)；根据Jaccard相似性系数原理，当C介于0.00~0.25 时为极不相似，C介于0.25~0.50时为中等不相似，C介于0.50~0.75时为中等程度相似，C介于0.75~1.00 时为极度相似[16]。由此可见，野生铁皮石斛根、茎、叶3个部位两两之间的内生细菌类群组成均为中等相似。
表 1 铁皮石斛内生细菌的多样性1)Table 1. The diversity of endophytic bacteria in Dendrobium officinale生境
Habitat植株部位
Plant part物种数
No. of bacteria
species多样性指数
Shannon-Wiener
diversity index (H′)均匀度指数
Evenness
index (E)相似性系数
Similarity coefficient (C)根 Root 茎 Stem 叶 Leaf 野生
Wild根 Root 6 0.75b 0.42b 0.72 0.60 茎 Stem 9 0.80b 0.36b 0.72 0.65 叶 Leaf 5 1.02a 0.63a 0.60 0.65 整株 Whole plant 9 0.85 0.39 人工栽培
Artificial cultivation根 Root 2 0.47b 0.68b 0.44 0.70 茎 Stem 3 0.87a 0.80a 0.44 0.64 叶 Leaf 3 0.93a 0.84a 0.70 0.64 整株 Whole plant 3 0.61 0.56 1)相同生境同列数据后的不同小写字母表示植株不同部位间差异显著(P<0.05，Duncan’s法)
1)Different lowercase letters in the same column of the same habitat indicate significant differences among different plant parts (P<0.05, Duncan’s test)人工栽培铁皮石斛内生细菌的H′为0.61，根、茎、叶的内生细菌H′分别为0.47、0.87、0.93，根、茎、叶内生细菌的E分别为0.68、0.80、0.84，说明叶中的内生细菌多样性最高，且类群最均匀，根的内生细菌H′和E显著低于茎和叶的相关指数；人工栽培植株中根与叶相似性最高(C根−叶=0.70)，其次是茎与叶(C叶−茎=0.64)，根与茎中最低(C根−茎=0.44)；根据Jaccard相似性系数原理判断标准，人工栽培铁皮石斛植株内生细菌类群在根−叶和茎−叶中均为中度相似，根−茎中为中度不相似。
2.3 野生铁皮石斛活性内生菌的筛选
以野生铁皮石斛代表性内生细菌为供试菌株，进行解磷、解钾、固氮、产铁载体、产IAA、拮抗菌的活性筛选，共获得38株菌株，占筛选菌株的45%，其中10株具有解钾活性，30株具有固氮活性，2株具有产IAA活性，4株具有拮抗病原菌活性。这些活性菌株中，10株分离自根，25株分离自茎，3株分离自叶，分子生物学鉴定归类为芽孢杆菌属(28株)、无色菌属(3株)、不动杆菌属(1株)、短小杆菌属(1株)、硫胺素芽孢杆菌属(2株)、泛菌属(1株)、短食单胞菌属(1株)、寡养单胞菌属(1株)。菌株Bacillus sp.B70、Curtobacterim sp.B81同时具有固氮和产IAA活性。菌株Bacillus sp.B03、Bacillus sp.B23、Stenotrophomons sp.B32、Bacillus sp.B41、Achromobacter sp.B68、Bacillus sp.B83同时具有解钾和固氮的特性。说明野生铁皮石斛内生细菌具备较好的促生潜力，是发展菌剂的良好备选。
2.4 野生铁皮石斛内生细菌在铁皮石斛组培苗中的定殖
菌株Bacillus sp.B70、Curtobacterim sp.B81是促生活性较高的菌株，不动杆菌属Acinetobacter是野生铁皮石斛优势菌群，泛菌属Pantoea细菌在野生和人工栽培铁皮石斛中都存在，菌株Acinetobacter sp.B48和Pantoea sp.B28具有拮抗病原菌活性。以菌株Bacillus sp.B70、Curtobacterim sp.B81、Acinetobacter sp.B48 和Pantoea sp.B28为供试菌，观察它们在铁皮石斛组培苗的定殖动态。结果表明，除了Curtobacterim sp.B81以外，其他3株菌株能定殖在组培苗的不同部位(表2)。菌株Pantoea sp.B28、Acinetobacter sp.B48、Bacillus sp.B70在回接3 d后能在植物宿主中分离到，说明内生菌具有一定的定殖效率。在回接后10 d，内生菌定殖部位和数量有所增多，是内生菌定殖的发展时期。回接后20 d，定殖部位有所减少，说明内生菌定殖因组织部位环境不同出现偏好性。定殖菌回接后30与20 d相比，除Acinetobacter sp.B48在叶中的定殖消失，其余菌株定殖部位相对稳定，是定殖相对稳定期。在30 d的定殖观察中，菌株Acinetobacter sp.B48和Bacillus sp.B70最终定殖在茎部，菌株Pantoea sp.B28能定殖在根、茎、叶各部位。
表 2 内生菌在铁皮石斛组培苗中的定殖动态1)Table 2. Colonization trends of endophytic bacteria in potted seedling of Dendrobium officinale回接菌株
Backgrafted strain回接后3 d
Three days after backgrafting回接后10 d
10 days after backgrafting回接后20 d
20 days after backgrafting回接后30 d
30 days after backgrafting根 Root 茎 Stem 叶 Leaf 根 Root 茎 Stem 叶 Leaf 根 Root 茎 Stem 叶 Leaf 根 Root 茎 Stem 叶Leaf Pantoea sp.B28 − + − + + + + + + + + + Acinetobacter sp.B48 − − + + + + − + + − + − Bacillus sp.B70 + + + + + + − + − − + − Curtobacterim sp.B81 − − − − − − − − − − − − 1)“−”表示没有分离到回接菌，“+”表示分离到回接菌
1) “−”denotes no isolation of inoculated bacteria; “+”denotes isolation of inoculated bacteria3. 讨论与结论
兰科植物有着丰富的内生细菌资源，已有研究报道其在根、茎和叶中均有分布，对宿主植物的生长、抗病和抗逆性等具有重要作用[17]。本研究从铁皮石斛根、茎、叶中分离到285株内生细菌，隶属3门11属。从野生铁皮石斛共分离到217株内生细菌，归类为芽孢杆菌属、微杆菌属等9个属，以芽孢杆菌属、不动杆菌属为优势菌群存在于根、茎、叶各部位。从人工栽培铁皮石斛植株中只分离到68株内生细菌，归类为伯克霍尔德菌属、埃希菌属、泛菌属3个属，以伯克霍尔德菌属、埃希菌属为优势菌群存在于植株根、茎部。只有泛菌属在野生和人工栽培铁皮石斛中都有分布，野生铁皮石斛内生细菌物种多样性指数均明显高于人工栽培铁皮石斛内生细菌，表明两者内生细菌组成结构存在较大差异。这可能是因为野生铁皮石斛多生长在物种多样的山林环境，该环境蕴含了丰富的微生物资源，而人工种植大棚环境单一，物种有限，栽培基质频繁消毒处理，长期的人工栽培模式大大降低了植物内生菌的多样性。植物缺乏内生菌也是其生长缓慢、成活率低、抗逆性差的主要因素[10]。把野生铁皮石斛中具有促生和生防活性的内生菌接入人工栽培铁皮石斛，一方面可提高人工栽培铁皮石斛内生菌多样性，增强其对栽培环境的适应性，另一方面也为铁皮石斛仿生态栽培奠定基础。
钾和磷是植物生长必需的营养元素。IAA可以促进根的生长，增强根毛增殖和延长。固氮菌为宿主提供一定氮源，满足其生命需求，有利于增加产量。产铁载体的内生细菌可以螯合Fe3+，有效地竞争致病真菌对铁的需求[14]。炭疽病是人工栽培铁皮石斛的重要病害，严重影响石斛产量和品质[15]。从野生铁皮石斛内生细菌中筛选解磷、解钾、固氮、产铁载体、产IAA、拮抗炭疽菌活性菌株，共获得38株活性菌株，占筛选菌株的45%，这些内生细菌具有开发促生和生防菌剂的良好潜力。但是菌剂应用在生产栽培实践中面临的主要问题是有效期短和效果不稳定[18]，其主要原因是活性菌在自然条件下在宿主植物的定殖能力有限。因此定殖能力强是内生菌开发为生产应用菌剂的前提条件。本研究将促生活性较高的菌株Bacillus sp.B70、Curtobacterim sp.B81及优势类群的菌株Acinetobacter sp.B48、Pantoea sp.B28回接组培苗，除了Curtobacterim sp.B81以外，其余菌株在组培苗都有定殖分布，说明来源于野生铁皮石斛的内生菌在人工组培苗中仍有较好的定殖性。虽然是通过根部接种，但是菌株Bacillus sp.B70在回接后20 d只存在于茎部，菌株Acinetobacter sp.B48存在于茎和叶部，这可能是因为植物不同组织部位结构和成分不同，内生细菌在宿主植物中分布具有组织偏嗜性。
综上所述，铁皮石斛内生细菌资源丰富，野生和人工铁皮石斛内生细菌多样性有较大差异，内生细菌活性比较普遍，在人工组培苗中有一定的定殖性，这为铁皮石斛内生细菌功能性开发和利用奠定了基础。
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图 1 侵染细菌域和古菌域的dsDNA噬菌体分类及形态示意图
* 未分类 Unclassifed
Figure 1. Classification and morphological diagrams of dsDNA phages infecting bacterial and archaeal domains
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图 2 土壤噬菌体洗脱、富集、核酸提取流程图
Figure 2. Flowchart of elution, enrichment and nucleic acid extraction of soil phages
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图 4 噬菌体调控土壤微生物概念模型
Figure 4. The conceptual model for regulating soil microbes by phages
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